Chalcone/1,3,4-Oxadiazole/Benzimidazole hybrids as novel anti-proliferative agents inducing apoptosis and inhibiting EGFR & BRAFV600E

Introduction One of the most robust global challenges and difficulties in the 21st century is cancer. Treating cancer is a goal which continues to motivate researchers to innovate in design and development of new treatments to help battle the disease. Objectives Our objective was developing new antiapoptotic hybrids based on biologically active heterocyclic motifs "benzimidazole?oxadiazole-chalcone hybrids'' that had shown promising ability to inhibit EGFR and induce apoptosis. We expected these scaffolds to display anticancer activity via inhibition of BRAF, EGFR, and Bcl-2 and induction of apoptosis through activation of caspases. Methods The new hybrids 7a-x were evaluated for their anti-proliferative, EGFR & BRAFV600E inhibitory, and apoptosis induction activities were detected. Docking study & dynamic stimulation into EGFR and BRAFV600E were studied. Results All hybrids exhibited remarkable cell growth inhibition on the four tested cell lines with IC50 ranging from 0.95 μM to 12.50 μM. which was comparable to Doxorubicin. Compounds 7k-m had the most potent EGFR inhibitory activity. While, compounds 7e, 7g, 7k and 7l showed good inhibitory activities against BRAFV600E. Furthermore, Compounds 7k, 7l, and 7m increased Caspases 3,8 & 9, Cytochrome C and Bax levels and decreased Bcl-2 protein levels. Compounds 7k-m received the best binding scores and showed binding modes that were almost identical to each other and comparable with that of the co-crystalized Erlotinib in EGFR and BRAF active sites. Conclusion Compounds 7k-m could be used as potential apoptotic anti-proliferative agents upon further optimization. Supplementary Information The online version contains supplementary material available at 10.1186/s13065-023-01003-3.


Introduction
Treating cancer is a goal which continues to motivate researchers to innovate in design and development of new treatments to help battle the disease.Despite these efforts to combat cancer it is spreading quickly to all age groups and it is the second leading cause of death [1].The global burden of cancer is estimated to climb to 28.4 million in 2040, representing a 47% increase over 2020 [2].Lung, liver, stomach, breast, and colon cancer were the top five causes of cancer-related mortality worldwide [3].Lung and breast cancers alone represent nearly onefourth of all newly diagnosed cancer patients in 2020 [4 with lung cancer attributing to 18% of cancer mortality and breast cancer as the main cause of cancer mortality among women [5].
Apoptosis is an intriguing target for development of innovative cancer therapeutics.Normally, the apoptotic pathway becomes activated by DNA damage or uncontrolled proliferation [6].There are two pathways that activate apoptosis: the intrinsic and extrinsic pathways which correlate with intracellular and extracellular signals [7].The intrinsic pathway is activated in response to DNA damage and cytokine deprivation, whereas the extrinsic pathway is triggered by the immune system.The two pathways converge at the executioner caspases [3,6,7] which are a class of cysteine proteases that cleave target proteins required for normal cell function [8].Activation of caspases results in plasma membrane changes and shrinking of apoptotic cells that eventually lead to cell death [9].The intrinsic pathway is regulated by B-cell lymphoma-2 (Bcl-2) protein family which include proapoptotic effector proteins, proapoptotic BH3-only proteins, and antiapoptotic Bcl-2 proteins.Bcl-2 proteins inhibit apoptosis through inhibition of the proapoptotic Bcl-2 proteins, Bcl-2-associated X protein (BAX) and Bcl-2 homologous antagonist killer (BAK) [10].BH3-only proteins inhibit the antiapoptotic Bcl-2 proteins [11].
Apoptosis evasion is essential for survival of cancer cells and leads to increasing invasiveness, stimulating angiogenesis, deregulation of cell proliferation, and interference with differentiation.Usually, the predominant methods of apoptosis evasion include inhibition of intrinsic pathway and caspase function, overexpression of antiapoptotic Bcl-2 proteins, and loss of BAX and/or BAK [12].This results in resistance to any intrinsic apoptotic stimuli which includes some anticancer drugs [13].
Epidermal growth factor receptor (EGFR) and B-RAF play a role in regulating the mitogen-activated protein (MAP) kinase pathway which affects cell division, differentiation, and could lead to abnormal cell proliferation [14].Signaling pathways of EGFR control angiogenesis, activation, and regulation of cellular proliferation.Overexpression of the EGFR gene and mutations of the EGFR tyrosine kinase domain were reported in the development and progression of different carcinomas including lung, colorectal, breast, brain, and pancreas [15].Therefore these genetic alterations showed high probability to respond to EGFR small molecule tyrosine kinase inhibitors (TKI).Additionally, a recent study has shown that suppression of EGFR signaling was correlated to induction of intrinsic apoptosis in sensitive non-small cell lung cancer (NSCLC) EGFR-mutant cell lines [16].However, EGFR inhibitors have exhibited limited efficacies and have been challenged by innate and acquired resistance in the clinic [17].
Erlotinib, is one of the most popular EGFR inhibitors which is currently marketed to treat many types of cancer with EGFR gene mutations (including non-small lung and pancreatic cancer) [18].Its anticancer activity drives from suppressing intracellular phosphorylation of TK at the ATP binding site of the receptor, inhibiting JAK2V617F; a mutant version of JAK2 and inducing apoptotic cell death pathways [19].Despite its high potency, selectivity, and acceptable safety profile, patients rapidly develop resistance within 8-12 months from the start of treatment via mutation in the ATP binding pocket of the EGFR kinase domain [20].
EGFR signaling is a part of a complex network that has been the target of effective cancer therapies.However, emergence of resistance is a major hurdle to developing an effective anticancer regimen [21].The combination of an EGFR inhibitor and a BRAF inhibitor synergizes and improves anticancer activity through induction of apoptosis, as demonstrated by the combination of dabrafenib with cetuximab in colorectal cells [22].Therefore, combination therapy that combines an anti-EGFR, anti-BRAF, and apoptotic agent may exhibit a multi-pronged approach that can be developed into a highly attractive and specific molecular oriented remedy.
In our continued research efforts to develop antiapoptotic hybrids based on biologically active heterocyclic motifs we have discovered that benzimidazole-oxadiazole-chalcone hybrids have shown promising ability to inhibit EGFR and induce apoptosis.[23][24][25].Individually each motif has been reported as an effective anticancer scaffold with mechanisms of action as either EGFR inhibitor, BRAF inhibitor, or inducer of apoptosis [24,26,27].In this study we explored new chemical architectures attaching the benzimidazole directly to oxadiazole and chalcone.In these compounds variably substituted aromatic rings were attached to the 2-position of benzimidazole.We envisioned this structural modification would afford improved compounds capable of interacting with EGFR and stimulating caspase-3 to induce apoptosis.
Based on the previous silico molecular docking simulations using MOE software for the most active compounds and Erlotinib into ATP binding sites it was found that Erlotinib forms 2 H-bond interactions with Met769 and Gln767 and one cation…π interaction with Leu 820.None of the earlier benzimidazole-chalcone hybrids showed identical binding interactions as Erlotinib with the same amino acid residues.Moreover, the most potent EGFR inhibitors had one H-bonding interaction with Met769 as Erlotinib which meant this interaction was essential for activity.The new design moved the aromatic ring to a distal substitution on benzimidazole to introduce more flexibility in the structure.This modification allows the benzimidazole ring to be in closer proximity to more amino acids which are important for interaction with Erlotinib.
Herein, we report the design, synthesis and various biological evaluations of hybrid molecules formed from benzimidazole, oxadiazole, and chalcone derivatives aimed to improve anticancer activity by synergetic effect, decrease side effects, and minimize emergence of drug resistance.We expected these scaffolds to display anticancer activity via inhibition of BRAF, EGFR, and Bcl-2 and induction of apoptosis through activation of caspases.We present the synthetic strategy for preparing twenty-four new compounds, the results of detailed anticancer screening, and molecular docking.Lastly, mechanistic investigations of the most active compounds 7k, 7L, and 7m regarding EGFR and BRAF inhibition and induction of caspases are described.

Chemistry
The series of chemical reactions that were employed to prepare targeted benzimidazole-chalcone hybrids 7a-x is illustrated in Fig. 1.First, substituted 2-phenyl-1 H-benzo [d]imidazole-5-carboxylic acids 1a-e were synthesized through condensation of 3,4-diaminobenzoic acid with the appropriate aldehyde adduct in the presence of DMF for 3-6 h [28][29].Fisher esterification of the resulting benzimidazole carboxylic acids 1a-e with absolute ethanol using catalytic amount of concentrated H 2 SO 4 as a dehydrating agent with heating for 20 h at reflux afforded the corresponding ethyl ester compounds 2a-e [30].Carbohydrazide compounds 3a-e were prepared by heating the esters 2a-e with hydrazine mono hydrate for 6-7 h at reflux [30].Consequently, the carbohydrazide compounds 3a-e were converted to 1,3,4 oxadiazole-benzimidazole derivatives 4a-e by heating with carbon disulfide and KOH in absolute ethanol overnight [23,29].
Chalcone intermediates 5a-e were prepared by Claisen Schmidt condensation in the presence of KOH.A series of 2-bromo-N-(4-cinnamoylphenyl)acetamide derivatives [30].6a-e were synthesized by reaction of the appropriate chalcone with bromoacetyl bromide at 0 o C in a biphasic medium (DCM/water) using K 2 CO 3 as a base [30].Finally, the alkylation reaction that affords target compounds 7a-x was accomplished by coupling the oxadiazole and acetylated chalcone in the presence triethylamine and acetonitrile as solvent in good yields ranging from 73-89% [30].The structure of the new compounds was confirmed via NMR and mass spectral analyses. 1H NMR spectra of compounds showed singlet signal or two singlets due to rotamers at δ: 4.14-4.46ppm related to (S-CH 2 -CO).On other hand, in all compounds, the two protons of chalcone appear in the aromatic region as two doublet signals at δ: 7.56-7.74ppm and δ: 7.80-8.16ppm with coupling constant range J = 15.2-15.6.Additionally, the amide proton of the linker NH appears as singlet signal at δ: 10.83-11.16ppm and benzimidazole proton NH appears as singlet signal at δ: 13.10-13.48ppm. 13C NMR spectra of compounds showed two carbonyl groups related to C = O of chalcone and N-C = O appeared at δ: 186.96-189.12ppm and 160.22-169.50ppm, furthermore, SCH 2 appears at δ: 43.63-47.58ppm.

Anti-proliferative assays
In vitro one-dose anti-proliferative screening in NCI The twenty-four synthesized compounds were submitted to the National Cancer Institute (NCI), USA.Eighteen hybrids 7a-h, 7k-r, 7u, and 7v were selected by NCI for one-dose assessment of their anti-proliferative activities at 10 µM dose by Sulforhodamine B colorimetric assay.The assessment was performed in 60 cell lines derived from nine tumor subpanels, comprising lung, leukemia, melanoma, colon, renal, CNS, prostate, breast, and ovarian cancer cell lines.

In vitro anti-proliferative assays in Panc-1, A-549, MCF-7, and HT-29 cells
The propidium iodide (PI) tests 31, 32 were further used for assessment of the IC 50 of all synthesized compounds and compared to Doxorubicin as a positive control against four cancer cell lines; pancreatic cancer cell line (Panc-1), epithelial line cancer cell (A-549), breast cancer cell line (MCF-7), and colon cancer cell line (HT-29).
As presented in Table 2, all tested hybrids exhibited remarkable cell growth inhibition on the four tested cell lines with IC 50 ranging from 0.95 µM to 12.50 µM which was comparable to Doxorubicin (IC 50 ranging from 0.90 µM to 1.41 µM).The most effective compound was 7k with IC 50 ranging from 0.95 µM to 1.40 µM and the second most active one was 7L with IC 50 ranging from 1.10 µM to 1.65 µM.It was observed that the highest activity was displayed by compounds; 7k, 7L, and 7m in which R 1 = 4-OCH 3 .Additionally, these hybrids showed similar effects on the Panc-1, A-549, and HT-29 cell lines with slight variation in IC 50 values.Our structure-activity relationship (SAR) analysis revealed that the anticancer activity is reliant on pattern of substitution in both phenyl rings (Fig. 3).Anticancer activity was correlated to substitution on phenyl ring attached to position two of benzimidazole ring with an effective substitution order of p-methoxy group > p-chlorine atom >>> 3,4-dimethoxy groups.Further, it was noted that for optimal activity with p-chlorine on phenyl ring the substitution order on the chalcone phenyl ring was p-chlorine > p-methoxy >>> H.

In vitro toxicity assay in normal cells (cell viability assay)
To check the safety of prepared compounds and evaluate their selectivity towards tumor cells over normal ones, the toxicity of compounds 7a-x were evaluated by 3-(4,5-Dimethylthiazole-2-yl)-2,5-diphenyltetrazol (MTT) assay on the normal human mammary epithelial cell line (MCF-10 A) [33,34].The human mammary epithelial cell lines were incubated for four days with a concentration of 50 µM of compounds 7a-x.All compounds showed cell viability of more than 80% (Table 2.).The cytotoxic activity of 7k against MCF-7 was 96-fold higher than its toxicity against MCF-10 A. Similarly compounds 7L and 7v were 111 and 97-fold more selective towards LOX-IMVI cell line respectively.

EGFR inhibitory activity assay
To explore the molecular mechanism of the synthesized hybrids, an assessment of the EGFR inhibitory ability of the most potent ten compounds was performed using the EGFR-TK assay [35,36].The most active compounds; 7e, 7g, 7h, 7k-n, 7p, 7q, and 7v were selected to determine their enzymatic inhibitory activity and compared to Erlotinib and the findings are included in Table 3.The results showed that the tested compounds inhibited EGFR with IC 50 values ranging from 0.55 µM to 3.90 µM.The most Fig. 3 The effect of compounds derivatization on anti-proliferative activity effective compound 7k (R 1 = 4-OCH 3 , R 2 = H) demonstrated the greatest inhibitory efficacy against EGFR with an IC 50 of 0.55 µM, being approximately 7-fold less potent than Erlotinib (IC 50 = 0.08 µM).Compounds 7L and 7m rank second and third in activity, with IC 50 values of 0.80 µM and 0.90 µM, respectively.The remaining compounds displayed weak inhibitory activity against EGFR, with IC 50 values ranging from 1.15 µM to 3.90 µM.Consequently, compounds 7k, 7L, and 7m were considered promising agents which could be used as potential anti-proliferative agents targeting EGFR-TK after optimization.

Apoptosis induction activity
Caspases assays The effects of the most active compounds; 7k, 7l, and 7m on caspases (3, 8, and 9) were evaluated and compared to Doxorubicin treated and untreated cells [38].The results showed that the tested compounds increased the level of active caspase-3 by 7-8 folds when compared to control cells; and that compounds 7k, 7l, and 7m induced remarkable overexpression of caspase-3 protein level (501.60 ± 4.00, 492.50 ± 4.00 and 473.60 ± 3.50 pg/mL, respectively) and were comparable to Doxorubicin (503.50 ± 4.50 pg/mL).Compared to the untreated cells, the most active anti-proliferative derivative 7k showed an 8-fold increase in caspase-3 level.The effect of compounds 7k, 7l, and 7m on caspases 8 and 9 were also investigated to underline the role of intrinsic and extrinsic apoptotic pathways in these compounds' anti-proliferative actions.As shown in Table 4, compound 7k increased caspase 8 and 9 levels in comparison to untreated cells by 10 and 17 folds, respectively.Compound 7l increased caspase 8 and 9 levels by 9 and 16 folds, respectively.These results indicated that the new hybrids were capable of activating both the intrinsic and extrinsic pathways, with a stronger impact on the intrinsic pathway because caspase 9 levels were higher.

Cytochrome C assay
The amount of cytochrome C in the cell is critical for caspase activation and the beginning of the intrinsic apoptosis process [31].The results of testing compounds 7k, 7l, and 7m as Cytochrome C activators were shown in Table 4. Compounds 7k, 7l, and 7m increased Cytochrome C levels by 14, 13, and 11 times, respectively, when compared to untreated control cells.These results support the assumption that Cytochrome C overexpression and the activation of intrinsic apoptotic pathway were the causes of apoptosis.

Expression levels of BAX and Bcl-2 proteins
The effects of compounds 7k, 7l, and 7m on BAX and Bcl-2 protein levels in the MCF-7 breast cancer cell lines were studied further using Doxorubicin as a reference for comparison [39].The results in Table 5 showed that as compared to  Doxorubicin, the compounds 7k, 7l, and 7m caused a significant increase in BAX levels.Compound 7k induction of BAX level (279.50 pg/mL) was similar to Doxorubicin (276 pg/mL) with a 33-fold increase over control untreated cancer cell, followed by compound 7l (271 pg/ mL and 32-fold rise).Finally, compound 7k reduced antiapoptotic Bcl-2 protein levels in MCF-7 cells to 0.94 ng/ mL, followed by compound 7l (0.97 ng/mL) in comparison to Doxorubicin (0.98 ng/mL).

Cell cycle analysis and apoptosis detection
Cell cycle analysis Compound 7k was applied to MCF-7 cancer cell line with IC 50 concentration (0.95 µM) to assess its effect on cell cycle progression and apoptosis induction.Data obtained from cell cycle analysis (Fig. 4) revealed that 7k significantly increased the percentage of accumulation of MCF-7 cells in G0-G1 and S phase 61.95% and 34.86% respectively as compared to 58.27% and 29.51% in untreated cells.On other hand, incubation with 7k decreased percentage accumulation in the G2/M phase to 3.19% versus 12.22% in control.From the previous results, it was concluded that compound 7k might arrest cells at G1/S and prevent it from entering G2/M phase as indicated by decreased percentage of accumulation of cells in G2/M phase compared to control.

Apoptosis detection
To further study the root for cytotoxic activity of compound 7k, Annexin V-FITC/PI assay method 40, 41 was used to investigate its capability to induce apoptosis.In this assay MCF-7 cell line was treated with IC 50 concentration (0.95 µM) of 7k and stained with Annexin V/PI then incubated for 24 h.Results showed that compound 7k increased the level of both early and late apoptosis to 28.71% and 12.63% respectively whereas necrosis level was only 3.05% (Figs. 5 and 6).These cumulative results further indicate that compound 7k showed potent anti-proliferative activity by induction of apoptosis.

Docking study into EGFR and BRAF V600E
The structures of the most bioactive derivatives (i.e., 7e, 7g, 7h, 7k-n, 7p, 7q, and 7v) were docked inside the active sites of EGFR and BRAF to investigate their binding interactions with these proteins (Figs. 7 and 8).The resulting docking scores of these derivatives were convergent (i.e., docking scores ranged from − 8.4 to -10.7 kcal/ mol, Table 6) and comparable with that of the co-crystalized ligands Erlotinib and vemurafenib, respectively.With regards to EGFR, compounds 7k, 7l, and 7m received the best binding scores (-10.6,-10.7, and − 10.4, respectively).Additionally, they showed binding modes that were almost identical to each other (RMSD = 0.79 Å) and comparable with that of the co-crystalized Erlotinib (docking score = -9.7 kcal/mol).
To further validate the docking results, the best docking poses for 7k, 7l, and 7m inside the kinase domains of both EGFR and BRAF were subjected to 100 ns-long MD simulations.As shown in Fig. 9, the structures of the three compounds (i.e., 7k, 7L, and 7m) exhibited significant stability inside the active sites of both enzymes with low deviations from their initial state (i.e., docked poses) (Average RMSDs ranged from 1.9 Å to 2.2 Å).Accordingly, the calculated absolute binding free energy (ΔG Bind ) for each compound using the MM-PBSA method were convergent and comparable with the co-crystalized inhibitors (Erlotinib and Vemurafenib, respectively) (Table 7) [42].Judging from the previous in silico structural analysis, it can be concluded that the derivatives 7k, 7L, and 7m are considered promising structure motifs acting as EGFR and BRAF dual inhibitors.

In silico prediction of physicochemical and pharmacokinetic properties
In this study, we used two web servers Swiss ADME (http://www.swissadme.ch/index.php)and PKCSM (http://biosig.unimelb.edu.au/pkcsm/) to investigate the physicochemical and pharmacokinetic features of compounds 7k, 7l and 7m.Swiss ADME affords information about SILICOS-IT, MLOGP, iLOGP, XLOGP3, and WLOGP, the distinct models that predict lipophilicity.Also, Log of Consensus Po/w is calculated by taking their arithmetic mean [43].BOILED Egg is a map of polarity expressed in TPSA, another model for predicting lipophilicity.The yolk reflects the potential for BBB permeability in the BOILED Egg plot (Fig. 10), whereas the white represents the possibility for GI absorption.Finally, a bioavailability radar map is a plot of six different physicochemical parameters: size, polarity, flexibility, solubility, saturation, and lipophilicity.The pink hexagon in the center of the figure represents the optimal range for excellent oral bioavailability (Fig. 11) [44,45].
PKCSM affords important information about pharmacokinetic parameters of the drug such as: Caco-2 permeability, volume of distribution at steady state (VDss), Pgp I and II inhibitors, total clearance, central nervous system (CNS) permeability, AMES toxicity, renal organic cation transporter 2 (OCT2) substrate, maximum recommended tolerated dose (MRTD) human, oral rat acute toxicity (LD 50 ) and chronic toxicity-lowest observed adverse effect (LOAEL), skin sensitization, hepatotoxicity, Tetrahymena pyriformis toxicity, and LC 50 by fathead minnow toxicity [46].SwissADME results of compounds 7k, 7l, 7m, and Erlotinib are presented in Table 8.These results indicated that compounds 7k and 7m obeyed Lipinski rule with one violation (M.wt > 500) while 7l did not; with two violations: M,wt > 500 and MLOGP of 4.17 (> 4.15).Compound 7k calculated octanol/water partition coefficient (log P) is 3.28 which is equal to Erlotinib while the log P values for 7l and 7m were 5.87 and 5.41, respectively.The three compounds showed low molecular flexibility (rotatable bond (RB) > 10) which may indicate poor absorption of the compounds.
All three compounds showed promising cell permeability through diverse biological membrane as topological polar surface area (TPSA) results ranged from 148.30 to 157.53 while Erlotinib was 74.73.PKCSM results were slightly different from Swis-sADME results.The difference in prediction outcomes between web servers are most likely due to changes in the modules and algorithms utilized on each web server.As a result, it is critical to receive ADMET characteristics from many web servers rather than just one [47].
The results shown in Table 9, revealed that the compounds 7k, 7l, and 7m displayed good values for oral absorption.Erlotinib was predicted to have a cellular permeability of 97.99% while compounds 7k, 7l, and 7m values ranged from 93.26 to 98.11%.The three compounds showed higher water solubility equal to -2.89 log mol/L than Erlotinib − 5.11 log mol/L.and displayed good skin permeability but low permeability in Caco2 (human colon adenocarcinoma).It was also predicted that 7k, 7l, and 7m may be a substrate of the ATP-binding cassette transporter P-glycoprotein, which was found to have an inhibitory effect in epithelial cells.Concerning distribution, the compounds showed low volume of distribution at a steady state at a value of -0.011, -0.009 and-0.038log L/kg which was comparable to Erlotinib value of 0.167 log L/kg.Distribution parameter revealed that the compounds were poorly distributed to blood brain barrier and displayed safety in CNS.It was predicted that the three compounds have no or little drug-drug interactions as they were shown to be CYP3A4 but not CYP2D6 substrates, and CYP2C19, CYP2C9, and CYP3A4 but not CYP2D6 inhibitors.
According to excretion parameters all compounds showed better results for total clearance from liver and kidney with values of 0.678, 0.669 and 0.703 log ml/min/ kg compared to Erlotinib with 0.572 log ml/min/kg value.Furthermore, the software predicted toxicity profiles including mutagenicity and cardiotoxicity.The three compounds and Erlotinib showed similar safety profile and acceptable level profile hERG I (human ether-a-gogo-related gene I) but displayed hepatotoxicity.Based on the in-silico ADME/Tox prediction, we may infer that compounds 7k, 7l, and 7m may possess an acceptable pharmacokinetic and safety profile, validating its promise as an oral therapy for cancer.Complete ADMET results of testing with PKCSM are presented in Table 9.

Conclusion
In this study we explored the apoptotic activity of new chemical architectures comprised of substituted benzimidazole moiety directly linked to an oxadiazole ring which is tethered to a chalcone derivative.The new hybrids' NCI assay results revealed that they had remarkable anti-proliferative activities, principally hybrids 7e, 7k, and 7m-o.Furthermore, in vitro anti-proliferative assays revealed promising inhibitory activity against a panel of four cancer cell lines, with IC 50 values comparable to Doxorubicin.Compounds 7k, 7l, and 7m displayed promising activity in activation of caspases, inhibiting EGFR and/or BRAF V600E , induction of BAX and inhibition of Bcl-2 proteins expression.Further docking, MD simulations, and ΔG Bind experiments were conducted to study the binding modes and stability of 7k, 7l, and 7m inside the kinase domains of both EGFR and BRAF.These results indicated that these compounds could be used as potential apoptotic anti-proliferative agents upon further optimization.ADMET properties of compounds 7k, 7l, and 7m were predicted successfully by two web servers Swiss ADME and PKCSM.The results revealed that the compounds had an ADMET profile that is comparatively similar to Erlotinib.

Chemistry
General Details: See Appendix A.

Table 7
The calculated ΔG Bind (in kcal/mol) of 7k, 7l, and 7m inside the kinase domains of both EGFR and BRAF (A and B, respectively) in comparison with the previously reported co-crystalized inhibitors (i.e., Erlotinib and Vemurafenib, respectively)

Fig. 6
Fig.6  Cell cycle and apoptosis induction analysis of MCF-7 cells after incubation with IC 50 of compound 7k (0.95 µM) for 24 h using Annexin V/PI compared to control untreated MCF-7

Fig. 8 A
Fig. 8 A: Binding modes of compounds 7k, 7l, 7m (green, cyan, and brick red structures, respectively) inside the binding site of kinase domain of human BRAF (PDB ID: 3OG7) B: Kinase domain of EGFR with the co-crystalized Erlotinib/Vemurafenib (orange-colored structure)

Table 3
Inhibitory activities of selected compounds against EGFR and BRAFV600E

Table 4
Effects of compound 7k, 7l, and 7m on caspases activity and amount of Cytochrome C in MCF-7 breast cancer cells

Table 5
Effects of compound 7k-m on the protein expression levels of BAX and Bcl-2

Table 9
ADMET Results of the test with PKCSM for the target compounds 7k, 7l, 7m and Erlotinib : 3OG7) using AutoDock Vina software program as reported in the literature.See Appendix A. ID